Ethanol dependence is a widespread affliction with societal costs in the hundreds of billions of dollars every year. Treatment of ethanol dependence is complicated by many factors, most notably relapse. Relapse behaviors are believed to result from ethanol withdrawal-induced anxiety (EWIA). A brain region critically involved in EWIA is the central amygdala (CeA). Lesions of the central, but not basolateral, amygdala resulted in reduced ethanol consumption during withdrawal. Neurobiological alterations induced by ethanol dependence are widespread;in the CeA, these changes include increased glutamatergic activity and NMDA receptor expression. During withdrawal, these increases lead to hyperactivation of the CeA;this hyperactivation is believed to mediate the withdrawal-induced anxiety that underlies relapse. Cocaine- and amphetamine-regulated transcript (CART) is a novel neuropeptide implicatied in ethanol dependence. It is expressed in the CeA and has been shown to increase expression here during acute withdrawal. Additionally, CART, has been reported to potentiate NMDA receptor-mediated currents. The overarching hypothesis of this proposal is that CART is a critical mediator of EWIA. To test this, a comprehensive examination of CART expression during dependence and withdrawal will be conducted. Ethanol dependence will-be induced using two models: chronic ethanol treatment (CET) and chronic intermittent ethanol (CIE). CIE contains a withdrawal component that presumably involves withdrawal induced anxiety. Dependence liability in wild type (WT) and CART knockout (KO) mice will compared using both CET and CIE. An examination of EWIA in the WT and KO mice at 0, 24, and 72 hours withdrawal will also be conducted. Corticosterone ELISAs and immunohistochemistry will be performed as well. Stress induced drinking and corticosterone levles in response to an acute stressor will also be assessed and compared between the WT and KO mice. The second half of this proposal will first verify in our hands, the NMDA potentiating actions of CART, specifically in the CeA. Then, a comprehensive electrophysiological voltage clamp analysis and comparison of NMDA receptor-mediated currents in the CeA of ethanol naive, CET, and CIE mice at 0, 24, and 72 hours withdrawal in WT and KO mice will be performed. The findings from these experiments will elucidate the underpinnings of EWIA and the role of CART in EWIA-induced relapse. Furthermore, these results will provide a novel target for therapeutic intervention and prevention of relapse in individuals dependent on ethanol and possibly other drugs of abuse.